The establishment of the primary body axis requires the sequential activities of two critical embryonic signaling centers: the anterior visceral endoderm (AVE) and node. The AVE specifies anterior character prior to overt morphological polarity in the embryo, whereas the node subsequently patterns the midline and posterior structures. Members of the TGFbeta superfamily have been implicated as essential intercellular mediators of AVE and node activities. These secreted factors transmit their signals through cell surface receptors and a recently identified group of at least nine intracellular proteins calls Smads. Of relevance to this proposal, Smad2 is specifically phosphorylated and activated by TGFbeta or activin ligand stimulation. To evaluate the role of TGFbeta/activin- related signaling in the developing embryo, the Robertson lab previously generated a null mutation in the Smad2 gene. Smad2 homozygous null embryos fail to establish the A-P axis and lack embryonic mesoderm. Interestingly, two conserved Smad2 isoforms are expressed in the early mouse embryo. Emerging biochemical data suggests that these two proteins are functionally distinct and likely to regulate independent subsets of target genes. I propose a molecular genetic approach that combines experiments in mouse and Xenopus to evaluate in vivo the individual roles of these two isoforms during axis specification and mesoderm formation in the mouse embryo. In addition, the definitive role of TGFbeta-related ligands in the node is largely unknown. I therefore plan to conditionally inactivate Smad2 to investigate the requirement for TGFbeta-related pathway in the formation and function of the node.